Method for orthogonally analyzing polypeptide impurity spectrum by using two-dimensional liquid chromatography technology

Abstract

The invention discloses a method for orthogonally analyzing a polypeptide impurity spectrum by using a two-dimensional liquid chromatography technology, which comprises the following steps: S1, carrying out one-dimensional separation on polypeptide in a sample by using an acid system reversed-phase chromatography, the acid system being composed of a TFA aqueous solution and a TFA acetonitrile solution; s2, performing two-dimensional orthogonal chromatographic analysis on the polypeptide obtained by one-dimensional separation through reversed phase chromatography of an alkaline system, wherein the alkaline system consists of an ammonium bicarbonate aqueous solution and acetonitrile. According to the detection method provided by the invention, impurities can be more effectively separated from a main peak, the impurity separation effect is improved, and data redundancy and impurity overlapping are reduced through screening and optimization of elution conditions, so that high-precision purity calculation is favorably realized, and the analysis efficiency and analysis precision of polypeptide impurities are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for orthogonally analyzing polypeptide impurity spectra using two-dimensional liquid chromatography technology. Background Art

[0002] Peptide impurities refer to non-target products or by-products produced during peptide synthesis, purification or storage. These impurities may affect the activity, stability and safety of the peptide, so they need to be strictly controlled. Peptide impurities mainly include missing peptides, inserted peptides, truncated peptides, protective group residues, aggregates, oxidation products, deamidation products and hydrolysis products.

[0003] The structures and physicochemical properties of many impurities are similar to those of the main components of peptides, which makes the analysis and separation of peptide impurities a complex task. The identification and separation of impurities are particularly important in biopharmaceuticals and the preparation of peptides with high purity requirements. In the prior art, commonly used methods for analyzing peptide impurities include reversed-phase high-performance liquid chromatography, ion exchange chromatography, size exclusion chromatography, capillary electrophoresis, mass spectrometry and nuclear magnetic resonance. However, these methods generally have the following disadvantages: 1) In reversed-phase high-performance liquid chromatography, some impurities, such as isomers, cannot be effectively separated from the main peak due to their similar properties to the main component, and the separation effect for polar impurities is also poor; 2) Ion exchange chromatography is suitable for separating charged impurities, but the separation effect for non-polar impurities is poor, and a complex buffer system is required, and there is also the possibility of affecting the stability of the peptide; 3) Size exclusion chromatography is suitable for separating aggregates, but the resolution is low and the separation effect for low molecular weight impurities is poor; 4) Capillary electrophoresis has high resolution and fast analysis speed, but it has the disadvantages of limited separation effect, poor reproducibility and high difficulty in operation; 5) Mass spectrometry has high sensitivity, but the ability to distinguish impurities is limited, and the operation is complex and the cost is high; 6) Nuclear magnetic resonance has low sensitivity, long time and expensive equipment. For reversed-phase high-performance liquid chromatography, in order to improve the separation effect, existing analytical schemes usually use two or more one-dimensional analytical methods to separate peptide impurities. However, although these methods can partially identify impurities, their results are prone to repeated separation, and it is impossible to effectively use the data of multiple one-dimensional analytical methods for accurate purity calculation.

[0004] In summary, there is an urgent need in the art for a peptide impurity analysis method with good separation, which can achieve high-precision purity calculation and improve analysis efficiency. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for orthogonally analyzing a polypeptide impurity profile using two-dimensional liquid chromatography technology, the method comprising:

[0006] S1. performing one-dimensional separation of the polypeptides in the sample by acidic system reverse phase chromatography, wherein the acidic system consists of a TFA aqueous solution and a TFA acetonitrile solution;

[0007] S2. Performing two-dimensional orthogonal chromatography analysis on the polypeptide obtained by one-dimensional separation by reverse phase chromatography using an alkaline system, wherein the alkaline system consists of an aqueous ammonium bicarbonate solution and acetonitrile.

[0008] Specifically, the polypeptide impurities include deleted peptides, inserted peptides, diastereomers, fragment peptides, deamidated impurities and amino acid modified impurities.

[0009] Specifically, the two-dimensional orthogonal chromatography analysis refers to dividing the main peak obtained by one-dimensional separation into 10 parts, and then introducing them into the two-dimensional orthogonal chromatography one by one through a switching valve for analysis.

[0010] Specifically, the liquid chromatography conditions for the one-dimensional separation are:

[0011] The chromatographic column is a phenyl-hexyl chromatographic column, and the preferred chromatographic column model is ACQUITY UPLC CSH Phenyl-Hexyl; the wavelength is 214 nm; the flow rate is 0.3 mL / min; the injection volume is 3 μL; the gradient elution program is:

[0012] Time(min) A％ B％ 0.0 90.0 10.0 1.0 90.0 10.0 16.0 75.0 25.0 30.0 50.0 50.0 30.1 10.0 90.0 35.0 10.0 90.0 35.1 90.0 10.0 40.0 90.0 10.0

[0013] Mobile phase A was 0.1% TFA in water, and mobile phase B was 0.1% TFA in acetonitrile.

[0014] Specifically, the liquid chromatography conditions of the two-dimensional orthogonal chromatography analysis are:

[0015] The chromatographic column is a phenyl-hexyl chromatographic column, and the preferred chromatographic column model is ACQUITY UPLC CSH Phenyl-Hexyl; the wavelength is 214 nm; the flow rate is 0.4 mL / min; the injection volume is 40 μL*80%*10; the gradient elution program is:

[0016]

[0017]

[0018] Mobile phase A was 20 mM (NH 4 ) 2 CO 3 The mobile phase B is acetonitrile solution.

[0019] Specifically, the switching time parameters of the switching valve are: high resolution, 2.65 seconds / cut, 10 cuts, single injection.

[0020] Specifically, the qualitative and quantitative analysis of the method includes the following steps:

[0021] 1) Infer the specific structure of impurities based on MS molecular weight;

[0022] 2) Calculate the total peak area of ​​each impurity using the following formula:

[0023]

[0024] S total =∑ j S j ;

[0025] 3) Normalize the peak area of ​​each impurity to obtain the percentage of the main peak. The calculation formula is as follows:

[0026]

[0027] 4) Calculate the percentage of each impurity in the sample;

[0028]

[0029] Among them, A ij : The peak area of ​​impurity j in the ith slice in the two-dimensional chromatogram;

[0030] S j : The sum of the peak areas of all slices of impurity j in the two-dimensional chromatogram;

[0031] S total : The sum of all impurity peak areas;

[0032] P j : The area percentage of impurity j in the main peak;

[0033] R: the total proportion of n slices of the main peak in the one-dimensional chromatogram, that is, the proportion of the main peak area to the total sample area;

[0034] C j : Final percentage of impurity j in the sample.

[0035] Specifically, an Agilent 1290UPLC ultra high performance liquid chromatography system was used for detection.

[0036] Specifically, the polypeptide sample to be tested is dissolved in 20% acetonitrile solution to prepare a 1 mg / mL solution.

[0037] Specifically, in S1, the first sample was run at least 3 times to ensure that the main peak time was stable before subsequent operations.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The method for orthogonally analyzing the impurity spectrum of a polypeptide using two-dimensional liquid chromatography technology provided by the present invention adopts a one-dimensional TFA reverse system combined with a two-dimensional ammonium bicarbonate alkaline system, combines the acid-base system, and utilizes different separation mechanisms to enable impurities to be more effectively separated from the main peak, thereby improving the impurity separation effect, and through the screening and optimization of elution conditions, reducing data redundancy and impurity overlap, thereby facilitating high-precision purity calculation and improving the analysis efficiency and analysis accuracy of polypeptide impurities.

[0040] 2. The method of orthogonally analyzing the impurity spectrum of a polypeptide using two-dimensional liquid chromatography technology provided by the present invention uses two-dimensional chromatography technology to cut the impurities in the main peak that cannot be separated in a one-dimensional chromatography system into a two-dimensional orthogonal system for separation, which can effectively separate the isomers and deamidation in the main peak that are difficult to separate by conventional methods. In addition, it also shows excellent separation effects for impurities such as missing peptides and inserted peptides.

[0041] 3. Compared with the prior art, the method for orthogonally analyzing the peptide impurity spectrum using two-dimensional liquid chromatography technology provided by the present invention can also achieve accurate quantitative calculation. By establishing a two-dimensional data fusion model, the accuracy of peptide impurity purity calculation is effectively improved, which has a significant and far-reaching impact on the qualitative and quantitative analysis of peptide impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 A schematic diagram of the method for orthogonal analysis of peptide impurity profiles using two-dimensional liquid chromatography provided in this application;

[0043] Figure 2 A schematic diagram for segmenting a chromatogram obtained by one-dimensional separation;

[0044] Figure 3 Schematic diagram of two-dimensional separation results. DETAILED DESCRIPTION

[0045] The technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The materials, instruments and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0046] Embodiment 1

[0047] In a specific embodiment, the peptide impurity spectrum in the sample to be tested is analyzed and detected using Agilent 1290UPLC with DAD detector and Agilent 1260UPLC with DAD detector. The specific operation is as follows:

[0048] 1. If Figure 1 As shown, the peptides in the sample are separated in one dimension by reverse phase chromatography using an acidic system composed of an aqueous TFA solution and a TFA acetonitrile solution, and then the peptides separated in one dimension are analyzed by two-dimensional orthogonal chromatography using reverse phase chromatography using an alkaline system composed of an aqueous ammonium bicarbonate solution and acetonitrile. The instruments and parameters used are shown in Table 1 below:

[0049] Table 1 Instrument parameters

[0050]

[0051] 2. Operation process:

[0052] The peptide sample was dissolved in 20% acetonitrile solution to prepare a 1 mg / mL solution. The Agilent 1290 2D-LC system was used. The first dimension used an acidic system of 0.1% TFA water and 0.1% TFA acetonitrile as the mobile phase. The watersACQUITY UPLC CSH Phenyl-Hexyl Column was used. The injection volume was 0.3 μL. The sample was run at least 3 times to ensure that the main peak time was stable. Then the time range of the peak cut was set, such as Figure 2 As shown, the main peak was cut into 10 parts and introduced into the second dimension for analysis one by one through the switching valve. The second dimension chromatogram used 20mM (NH4) 2 CO 3 in water, pH = 9.5 as mobile phase A, acetonitrile as mobile phase B, it is an alkaline system with orthogonal separation effect with the first dimension, and the chromatographic column is the same as the first dimension.

[0053] 3. Result analysis:

[0054] The results are as follows Figure 3 As shown in the figure, 10 slices of the main peak in the one-dimensional chromatogram separated multiple impurities in the two-dimensional orthogonal system. The specific structure of the impurity can be inferred based on the MS molecular weight, and then the peak areas of each impurity in different slices in the two-dimensional are added up, and then normalized to obtain the percentage of the peak area of ​​each impurity, and then multiplied by the percentage of the entire slice in one dimension to obtain the percentage of each impurity in the sample. The calculation steps are as follows:

[0055] 1) Infer the specific structure of impurities based on MS molecular weight;

[0056] 2) Calculate the total peak area of ​​each impurity using the following formula:

[0057]

[0058] S total =∑ j S j ;

[0059] 3) Normalize the peak area of ​​each impurity to obtain the percentage of the main peak. The calculation formula is as follows:

[0060]

[0061] 4) Calculate the percentage of each impurity in the sample;

[0062]

[0063] Among them, A ij : The peak area of ​​impurity j in the ith slice in the two-dimensional chromatogram;

[0064] S j : The sum of the peak areas of all slices of impurity j in the two-dimensional chromatogram;

[0065] S total : The sum of all impurity peak areas;

[0066] P j : The area percentage of impurity j in the main peak;

[0067] R: the total proportion of n slices of the main peak in the one-dimensional chromatogram, that is, the proportion of the main peak area to the total sample area;

[0068] c j : Final percentage of impurity j in the sample.

[0069] The calculation results are shown in Table 2 below:

[0070] Table 2 Experimental results

[0071] peak1 peak2 peak3 peak4 peak5 peak6 M / Z,Z=3 1432.766 1596.17 1577.161 1577.161 1577.495 1548.154 ΔM -433 +57 0 0 +1 -87 ID Uknown +Gly Main Isomer deamidation -Ser cut1 5.04 9.03 21.71 cut2 9.01 11.8 306.55 15.41 cut3 5.52 2623.06 34.06 cut4 2050.05 cut5 2164.87 cut6 244.99 cut7 104.83 4.06 cut8 63.75 11.47 4.1 cut9 22.368 cut10 26.55 Sj 19.57 20.83 7628.728 49.47 15.53 4.1 Pj% 0.25％ 0.27％ 98.58％ 0.64％ 0.20％ 0.05％ Cj% 0.24％ 0.26％ 93.95％ 0.61％ 0.19％ 0.05％

[0072] In summary, it can be seen that the method provided by the present invention uses two-dimensional chromatography technology to cut the impurities in the main peak that cannot be separated in a one-dimensional chromatography system into a two-dimensional orthogonal system for separation, and separates the isomers of the main peak, deamidation impurities and other impurities that are difficult to separate by conventional methods. It also has a good separation effect on deleted peptides and inserted peptides, and the content of impurities can also be quantitatively calculated, which has good practicality and application prospects.

[0073] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography, characterized in that: The method comprises: S1. performing one-dimensional separation of the polypeptides in the sample by acidic system reverse phase chromatography, wherein the acidic system consists of a TFA aqueous solution and a TFA acetonitrile solution; S2. Performing two-dimensional orthogonal chromatography analysis on the polypeptide obtained by one-dimensional separation by reverse phase chromatography using an alkaline system, wherein the alkaline system consists of an aqueous ammonium bicarbonate solution and acetonitrile.

2. The method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography according to claim 1, characterized in that: The polypeptide impurities include deleted peptides, inserted peptides, diastereomers, fragment peptides, deamidated impurities and amino acid modified impurities.

3. The method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography according to claim 1, characterized in that: The two-dimensional orthogonal chromatography analysis refers to dividing the main peak obtained by one-dimensional separation into 10 parts, and then introducing them into the two-dimensional orthogonal chromatography one by one through a switching valve for analysis.

4. The method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography according to claim 1, characterized in that: The liquid chromatography conditions for the one-dimensional separation are: The chromatographic column is a phenyl-hexyl chromatographic column, and the preferred chromatographic column model is ACQUITY UPLC CSH Phenyl-Hexyl; the wavelength is 214 nm; the flow rate is 0.3 mL / min; the injection volume is 3 μL; the gradient elution program is: Mobile phase A was 0.1% TFA in water, and mobile phase B was 0.1% TFA in acetonitrile.

5. The method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography according to claim 1, characterized in that: The liquid chromatography conditions of the two-dimensional orthogonal chromatography analysis are: The chromatographic column is a phenyl-hexyl chromatographic column, and the preferred chromatographic column model is ACQUITY UPLC CSH Phenyl-Hexyl; the wavelength is 214 nm; the flow rate is 0.4 mL / min; the injection volume is 40 μL*80%*10; the gradient elution program is: Mobile phase A was 20 mM (NH4)2CO3 aqueous solution, and mobile phase B was acetonitrile solution.

6. The method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography according to claim 3, characterized in that: The switching time parameters of the switching valve are: high resolution, 2.65 seconds / cut, 10 cuts, single injection.

7. The method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography according to claim 1, characterized in that: The qualitative and quantitative analysis of the method includes the following steps: 1) Infer the specific structure of impurities based on MS molecular weight; 2) Calculate the total peak area of ​​each impurity using the following formula: S total =∑ j S j ; 3) Normalize the peak area of ​​each impurity to obtain the percentage of the main peak. The calculation formula is as follows: 4) Calculate the percentage of each impurity in the sample; Among them, A ij : The peak area of ​​impurity j in the ith slice in the two-dimensional chromatogram; S j : The sum of the peak areas of all slices of impurity j in the two-dimensional chromatogram; S total : The sum of all impurity peak areas; P j : The area percentage of impurity j in the main peak; R: the total proportion of n slices of the main peak in the one-dimensional chromatogram, that is, the proportion of the main peak area to the total sample area; C j : Final percentage of impurity j in the sample.

8. The method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography according to claim 1, characterized in that: Agilent 1290 UPLC ultra-high performance liquid chromatography system was used for detection.

9. The method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography according to claim 1, characterized in that: The peptide sample to be tested was dissolved in 20% acetonitrile solution to prepare a 1 mg / mL solution.

10. The method for orthogonal analysis of polypeptide impurity profiles using two-dimensional liquid chromatography according to claim 1, characterized in that: In S1, the first sample was run at least 3 times to ensure that the main peak time was stable before subsequent operations.

Images